WO1998053062A1 - Homologue du recepteur edg-6 humain - Google Patents

Homologue du recepteur edg-6 humain Download PDF

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WO1998053062A1
WO1998053062A1 PCT/CA1998/000487 CA9800487W WO9853062A1 WO 1998053062 A1 WO1998053062 A1 WO 1998053062A1 CA 9800487 W CA9800487 W CA 9800487W WO 9853062 A1 WO9853062 A1 WO 9853062A1
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seq
nucleic acid
protein
molecule
sequence
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PCT/CA1998/000487
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Donald G. Munroe
Tejal B. Vyas
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Allelix Biopharmaceuticals, Inc.
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Priority to AU74223/98A priority Critical patent/AU7422398A/en
Priority to CA002290713A priority patent/CA2290713A1/fr
Publication of WO1998053062A1 publication Critical patent/WO1998053062A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention is in the field of molecular biology; more particularly, the present invention describes a nucleic acid sequence and an amino acid sequence for a novel human EDG-6 receptor homologue.
  • edg endothelial differentiation gene receptors
  • orphan receptors because their endogenous ligands are not known (for example see Hla, T. and Maciag, T. (1990) J. Biol. Chem. 265:9308-13; US patent 5,585,476).
  • LPA lysophospatidic acid
  • T7Gs transmembrane G protein coupled receptors
  • T7Gs are so named because of their seven hydrophobic domains which span the plasma membrane and form a bundle of antiparallel helices.
  • These transmembrane segments (TMS) are designated by roman numerals I- VII and account for structural and functional features of the receptor.
  • the bundle of helices forms a binding pocket; however, when the binding site must accommodate more bulky molecules, the extracellular N-terminal segment or one or more of the three extracellular loops participate in binding and in subsequent induction of conformational change in intracellular portions of the receptor.
  • the activated receptor interacts with an intracellular G-protein complex which mediates further intracellular signaling activities generally the production of second messengers such as cyclic AMP (cAMP), phospholipase C, inositol triphosphate or ion channel proteins.
  • cAMP cyclic AMP
  • phospholipase C phospholipase C
  • inositol triphosphate ion channel proteins
  • T7G receptors are expressed and activated during numerous developmental and disease processes. Identification of a novel T7G receptor provides the opportunity to diagnose or intervene in such processes, and the receptor can be used in screening assays to identify physiological or pharmaceutical molecules which trigger, prolong or inhibit its activity. SUMMARY OF THE INVENTION
  • the invention provides a unique nucleotide sequence which encodes a novel human EDG-6 receptor homologue (HEDG).
  • HEDG human EDG-6 receptor homologue
  • the nucleotide sequence encoding HEDG is designated hedg.
  • the invention provides an isolated nucleic acid molecule wherein the nucleic acid molecule encodes a polypeptide having an amino acid sequence as shown in SEQ. ID NO:2.
  • the invention provides an isolated nucleic acid molecule having a nucleotide sequence as shown in SEQ. ID NO: 1.
  • the invention provides a nucleic acid molecule which is anti-sense to the molecules indicated above.
  • the invention provides for expression vectors, probes and DNA constructs based on the polynucleotides mentioned above.
  • the invention provides for a purified polypeptide having the amino acid sequence as shown in SEQ. ID NO:2.
  • the invention also provides for antibodies specific to the above polypeptide.
  • the invention provides for methods of purifying and assaying polypeptides as indicated above.
  • the invention provides for transgenic animals which include the nucleotide sequence of the invention.
  • Figures IA and IB shows the alignment of the nucleic acid sequence (coding region of SEQ. ID NO: 1) and amino acid sequence (SEQ. ID NO:2) for HEDG.
  • Figure 2 displays the nucleic acid sequence (SEQ. ID NO:3) of a cDNA encoding HEDG.
  • HEDG refers to an EDG-6 receptor homologue in either naturally occurring or synthetic form and active fragments thereof which have the amino acid sequence of SEQ. ID NO:2.
  • the polypeptide HEDG is encoded by mRNAs transcribed from the cDNA, as designated by the lower case abbreviation, hedg, of SEQ. ID NO:l.
  • the novel human EDG-6 receptor homologue, HEDG was cloned and isolated from a human kidney proximal tubule cDNA library. It shows 52.9% identity to human edg-2 (WO 97/00952).
  • oligonucleotide is a stretch of nucleotide residues which has a sufficient number of bases to be used as an oligomer, amplimer or probe in a polymerase chain reaction (PCR). Oligonucleotides are prepared from genomic or cDNA sequence and are used to amplify, reveal or confirm the presence of a similar DNA or RNA in a particular cell or tissue. Oligonucleotides or oligomers comprise portions of a DNA sequence having at least about 10 nucleotides and as many as about 35 nucleotides, preferably about 25 nucleotides.
  • Probes may be derived from naturally occurring or recombinant single - or double - stranded nucleic acids or be chemically synthesized. They are useful in detecting the presence of identical or similar sequences.
  • a "portion” or “fragment” of a polynucleotide or nucleic acid comprises all or any part of the nucleotide sequence having fewer nucleotides than about 6 kb, preferably fewer than about 1 kb which can be used as a probe.
  • Such probes may be labeled with reporter molecules using nick translation, Klenow fill-in reaction, PCR or other methods well known in the art. After optimizing reaction conditions to eliminate false positives, nucleic acid probes may be used in Southern, Northern or in situ hybridizations to determine whether DNA or RNA encoding HEDG is present in a cell type, tissue, or organ.
  • Reporter molecules are those radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents which associate with, establish the presence of, and may allow quantification of a particular nucleotide or amino acid sequence.
  • Reporter nucleotide variants encoding HEDG may be synthesized by making use of the "redundancy in the genetic code.
  • Various codon substitutions such as the silent changes which produce specific restriction sites or codon usage-specific mutations, may be introduced to optimize cloning into a plasmid or viral vector or expression in a particular prokaryotic or eukaryotic host system, respectively.
  • Chimeric molecules may be constructed by introducing all or part of the nucleotide sequence of this invention into a vector containing additional nucleic acid sequence which might be expected to change any one (or more than one) of the following HEDG characteristics: cellular location, distribution, ligand-binding affinities, interchain affinities, degradation/turnover rate, signaling, etc.
  • HEDG neuropeptide derived neuropeptide
  • “Active” refers to those forms, fragments, or domains of any HEDG polypeptide which retain the biological and/or antigenic activities of any naturally occurring HEDG.
  • “Naturally occurring HEDG” refers to a polypeptide produced by cells which have not been genetically engineered and specifically contemplates various polypeptides arising from post-translational modifications including, but not limited to, alternative promoter usage, alternative splicing, alternative polyadenylation and RNA editing as well, as acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.
  • Derivative refers to those polypeptides which have been chemically modified by such techniques as ubiquitination, labeling (see above), pegylation (derivatization with polyethylene glycol), and chemical insertion or substitution of amino acids such as ornithine which do not normally occur in human proteins.
  • Recombinant polypeptide variant refers to any polypeptide which differs from naturally occurring HEDG by amino acid insertions, deletions and/or substitutions, created using recombinant DNA techniques. Guidance in determining which amino acid residues may be replaced, added or deleted without abolishing activities of interest may be found by comparing the sequence of HEDG with that of related polypeptides and minimizing the number of amino acid sequence changes made in highly conserved regions. Variants in which certain functional properties of HEDG are inactivated (i.e. signal transduction) may nonetheless retain other functional properties (e.g. ligand binding).
  • substitutions are conservative in nature when they result from replacing one amino acid with another having similar structural and/or chemical properties, such as the replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.
  • “Insertions” or “deletions” are typically in the range of about 1 to 5 amino acids. The variation allowed may be experimentally determined by producing the peptide synthetically or by systematically making insertions, deletions, or substitutions of nucleotides in the hedg sequence using recombinant DNA techniques.
  • a "signal or leader sequence” can be used, when desired, to direct the polypeptide through a membrane of a cell. Such a sequence may be naturally present on the polypeptides of the present invention or provided from heterologous sources by recombinant DNA techniques.
  • oligopeptide is a short stretch of amino acid residues and may be expressed from an oligonucleotide. It may be functionally equivalent to and the same length as (or considerably shorter than) a "fragment", "portion”, or “segment” of a polypeptide. Such sequences comprise a stretch of amino acid residues of at least about 5 amino acids and often about 17 or more amino acids, typically at least about 9 to 13 amino acids, and of sufficient length to display biological and/or antigenic activity.
  • a "chimeric" recombinant HEDG polypeptide refers to any polypeptide consisting of at least about 5 amino acids and often about 9 to 15 amino acids. Typically, such chimeric recombinants consist of 16 or more amino acids of the HEDG sequence cova ntly linked or expressed as a fusion protein with portions of one or more different, naturally-occurring or artificially created polypeptide sequences. Chimeric HEDG polypeptides are designed to alter the physical, biochemical, or functional properties of HEDG. "Inhibitor” is any substance which retards or prevents a chemical or physiological reaction or response. Common inhibitors include but are not limited to antisense molecules, antibodies, and antagonists.
  • Standard expression is a quantitative or qualitative measurement for comparison. It is based on a statistically appropriate number of normal samples and is created to use as a basis of comparison when performing diagnostic assays, running clinical trials, or following patient treatment profiles.
  • Animal as used herein may be defined to include human, domestic (cats dogs, etc.), agricultural (cows, horses, sheep, etc.) or test species (mouse, rat, rabbit, etc.).
  • the present invention provides a nucleotide sequence uniquely identifying a novel seven transmembrane receptor (T7G), human EDG-6 or HEDG. Based on the homology of T7G, human EDG-6 or HEDG.
  • HEDG HEDG to edg-2 it is likely that HEDG binds a ligand of the same chemical class.
  • Edg-2 specifically binds lysophosphatidic acid (LPA) which is a phospholipid.
  • LPA lysophosphatidic acid
  • Phospholipids have been demonstrated to be important regulators cell activity, including mitogenisis (Xu et al.
  • LPA elicits growth factor-like effects on cell prolyeration (Moolenar (1996) J. Biol. Chem, 270: 12949-12952) and cell migration
  • the ligand for HEDG is likely to be a biologically important regulator of cell activity, and therefore aberrant expression of HEDG is likely to be associated with chronic or acute disease states. Further, modulators of HEDG activity are likely to be useful in treatment or prevention of such disease states.
  • HEDG ligands are likely to be found among the phospholipid class of compounds. Therefore, preferably phospholipid molecules should be screened to identify HEDG ligands. More preferably, lysophospholipds should be screened. Even more preferably, lysoglycerophospholipids should be screened. Most preferably, lysophosphatidic acid (LPA), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), lysophosphatidylinositol (LPI), lysophosphatidylcholine (LPC), lyso-platelet activating factor (lyso-PAF) and phosphatidic acid should be screened.
  • LPE lysophosphatidic acid
  • LPE lysophosphatidylethanolamine
  • LPS lysophosphatidylserine
  • LPI lysophosphatidylinositol
  • LPA has now been confirmed as a ligand and agonist for HEDG.
  • a diagnostic test for excessive expression of HEDG can accelerate diagnosis and proper treatment of abnormal conditions caused by viral, bacterial or fungal infections; allergic responses; mechanical injury associated with trauma; hereditary diseases; lymphoma, leukemia or carcinoma; or other conditions which activate the genes of lymphoid or neuronal tissues.
  • the nucleotide sequences encoding HEDG (or their complement) have numerous applications in techniques known to those skilled in the art of molecular biology.
  • nucleotide sequences encoding HEDG disclosed herein are exemplary of known techniques and are not intended to limit their use in any technique known to a person of ordinary skill in the art.
  • nucleotide sequences disclosed herein may be used in molecular biology techniques that have not yet been developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, e.g., the triplet genetic code, specific base pair interactions, etc.
  • HEDG-encoding nucleotide sequences may be produced. Some of these will only bear minimal homology to the nucleotide sequence of the known and naturally occurring HEDG.
  • the invention has specifically contemplated each and every possible variation of nucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the nucleotide sequence of naturally occurring hedg, and all such variations are to be considered as being specifically disclosed.
  • nucleotide sequences which encode HEDG, its derivatives or its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring hedg under stringent conditions, it may be advantageous to produce nucleotide sequences encoding HEDG or its derivatives possessing a substantially different codon usage. Codons can be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic expression host in accordance with the frequency with which particular codons are utilized by the host.
  • RNA transcripts having more desirable properties such as a greater half-life, than transcripts produced from the naturally occurring sequence.
  • Nucleotide sequences encoding HEDG may be joined to a variety of other nucleotide sequences by means of well established recombinant DNA techniques (Sambrook J et al (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold
  • vectors of interest include expression vectors, replication vectors, probe generation vectors, sequencing vectors, etc.
  • vectors of interest may contain an origin of replication functional in at least one organism, convenient restriction endonuclease sensitive sites, and selectable markers for one or more host cell systems.
  • Another aspect of the subject invention is to provide for hedg-specific hybridization probes capable of hybridizing with naturally occurring nucleotide sequences encoding HEDG. Such probes may also be used for the detection of similar T7G encoding sequences and should preferably contain at least 40% nucleotide identity to hedg sequence.
  • the hybridization probes of the subject invention may be derived from the nucleotide sequence presented as SEQ. ID NO: l or from genomic sequences including promoter, enhancers or introns of the native gene.
  • Hybridization probes may be labeled by a variety of reporter molecules using techniques well known in the art.
  • the invention relates to nucleic acid sequences that hybridize with such HEDG encoding nucleic acid sequences under stringent conditions.
  • “Stringent conditions” refers to conditions that allow for the hybridization of substantially related nucleic acid sequences. For instance, such conditions will generally allow hybridization of a sequence with at least about 85% sequence identity, preferably with at least about 90% sequence identity, more preferably with at least about 95% sequence identity. Such hybridization conditions are described by Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, 1989. Hybridization conditions and probes can be adjusted in well-characterized ways to achieve selective hybridization of human-derived probes.
  • Nucleic acid molecules that will hybridize to HEDG encoding nucleic acid under stringent conditions can be identified functionally, using methods outlined above, or by using for example the hybridization rules reviewed in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, 1989.
  • examples of the uses for hybridization probes include: histochemical uses such as identifying tissues that express HEDG; measuring mRNA levels, for instance to identify a sample's tissue type or to identify cells that express abnormal levels of HEDG; and detecting polymorphisms in the HEDG.
  • RNA hybridization procedures are described in Maniatis et al. Molecular Cloning, a Laboratory Manual (Cold Spring Harbor Press, 1989).
  • PCR as described US Patent No's. 4,683,195; 4,800,195; and 4,965,188 provides additional uses for oligonucleotides based upon the nucleotide sequence which encodes HEDG.
  • probes used in PCR may be of recombinant origin, chemically synthesized, or a mixture of both.
  • Oligomers may comprise discrete nucleotide sequences employed under optimized conditions for identification of hedg in specific tissues or diagnostic use. The same two oligomers, a nested set of oligomers, or even a degenerate pool of oligomers may be employed under less stringent conditions for identification of closely related DNA's or RNA's.
  • PCR polymerase chain reaction
  • nucleic acid primers can be ligated to the nucleic acid sought to be amplified to provide the hybridization partner for one of the primers. In this way, only one of the primers need be based on the sequence of the nucleic acid sought to be amplified.
  • PCR methods of amplifying nucleic acid will utilize at least two primers.
  • One of these primers will be capable of hybridizing to a first strand of the nucleic acid to be amplified and of priming enzyme-driven nucleic acid synthesis in a first direction.
  • the other will be capable of hybridizing the reciprocal sequence of the first strand (if the sequence to be amplified is single stranded, this sequence will initially be hypothetical, but will be synthesized in the first amplification cycle) and of priming nucleic acid synthesis from that strand in the direction opposite the first direction and towards the site of hybridization for the first primer.
  • Conditions for conducting such amplifications particularly under preferred stringent hybridization conditions, are well known. See, for example, PCR Protocols, Cold Spring Harbor Press, 1991.
  • nucleic acid sequences encoding HEDG or HEDG derivatives into vectors for the production of mRNA probes.
  • vectors are known in the art, are commercially available and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerase as T7 or SP6 RNA polymerase and the appropriate reporter molecules.
  • RNA polymerase as T7 or SP6 RNA polymerase
  • reporter molecules the appropriate reporter molecules.
  • synthetic chemistry may be used to introduce mutations into the nucleotide sequence. Alternately, a portion of sequence in which a mutation is desired can be synthesized and recombined with longer portion of an existing genomic or recombinant sequence.
  • the nucleotide sequence for hedg can be used in an assay to detect inflammation or disease associated with abnormal levels of HEDG expression.
  • the cDN A can be labeled by methods known in the art, added to a fluid, cell or tissue sample from a patient, and incubated under hybridizing conditions. After an incubation period, the sample is washed with a compatible fluid which optionally contains a reporter molecule. After the compatible fluid is rinsed off, the reporter molecule is quantitated and compared with a standard as previously defined.
  • the nucleotide sequence for hedg can be used to construct hybridization probes for mapping the native gene. The gene may be mapped to a particular chromosome or to a specific region of a chromosome using well known mapping techniques.
  • chromosomal spreads Verma et al (1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York City
  • flow- sorted chromosomal preparations or artificial chromosome constructions such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial PI constructions or single chromosome cDNA libraries.
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • PI constructions single chromosome cDNA libraries.
  • In situ hybridization of chromosomal preparations and physical mapping techniques such as linkage analysis using established chromosomal markers are invaluable in extending genetic maps. Examples of genetic map data can be found in the yearly genome issue of Science (e.g. 1994, 265: 198 If). Often locating a gene on the chromosome of another mammalian species may reveal associated markers which can be used to help identify the analogous human chromosome.
  • New nucleotide sequences can be assigned to chromosomal subregions by physical mapping.
  • the mapping of new genes or nucleotide sequences provide useful landmarks for investigators searching for disease genes using positional cloning or other gene discovery techniques.
  • a disease or syndrome such as ataxia telangiectasia (AT)
  • AT ataxia telangiectasia
  • any sequences mapping to that area may represent or reveal genes for further investigation.
  • the nucleotide sequence of the subject invention may also be used to detect differences in gene sequence between normal and carrier or affected individuals.
  • Nucleotide sequences encoding hedg may be used to produce a purified oligo - or polypeptide using well known methods of recombinant DNA technology. Goeddel (1990, Gene Expression Technology, Methods and Enzymology, Vol. 185, Academic Press, San Diego CA) is one among many publications which teach expression of an isolated nucleotide sequence.
  • the oligopeptide may be expressed in a variety of host cells, either prokaryotic or eukaryotic. Host cells may be from the same species from which the nucleotide sequence was derived or from a different species.
  • HEDG oligonucleotide
  • Cells transformed with DNA encoding HEDG may be cultured under conditions suitable for the expression of T7Gs, their extracellular, transmembrane or intracellular domains and recovery of such peptides from cell culture.
  • HEDG (or any of its domains) produced by a recombinant cell may be secreted or may be contained intracellularly, depending on the particular genetic construction used. In general, it is more convenient to prepare recombinant proteins in secreted form. Purification steps vary with the production process and the particular protein produced.
  • an oligopeptide can be produced from a chimeric nucleotide sequence. This is accomplished by ligating the nucleotides from hedg or a desired portion of the polypeptide to a nucleic acid sequence encoding a polypeptide domain which will facilitate protein purification (Kroll D.J. et. al. (1993) DNA Cell Biol. 12:441-53). Chimeric receptors are useful in the purification of recombinant HEDG polypeptides, detection of interaction of recombinantly expressed HEDG receptors with ligands, and investigation of intracellular trafficking, signal transduction and interaction of HEDG with other intracellular proteins.
  • fragments of HEDG may be produced by direct peptide synthesis using solid-phase techniques (e.g. Stewart et. al. (1969) Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco CA; Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154). Automated synthesis may be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Foster City, CA) in accordance with the instructions provided by the manufacturer. Additionally, a particular portion of HEDG may be mutated during direct synthesis and combined with other parts of the peptide using chemical methods.
  • solid-phase techniques e.g. Stewart et. al. (1969) Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco CA; Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154. Automated synthesis may be achieved, for example, using Applied Biosystems 431 A
  • HEDG for antibody induction does not require biological activity: however, the protein must be antigenic.
  • Peptides used to induce specific antibodies may have an aa sequence consisting of at least five amino acids (aa), preferably at least 10 aa. They should mimic a portion of the aa sequence of the protein and may contain the entire aa sequence of a small naturally occurring molecule such as HEDG.
  • An antigenic portion of HEDG may be fused to another protein such as keyhole limpet hemocyanin, and the chimeric molecule used for antibody production.
  • Antibodies specific for HEDG may be produced by inoculation of an appropriate animal with the polypeptide or an antigenic fragment.
  • An antibody is specific for HEDG if it is produced against an epitope of the polypeptide and binds to at least part of the natural or recombinant protein.
  • Antibody production includes not only the stimulation of an immune response by injection into animals, but also analogous processes such as the production of synthetic antibodies, the screening of recombinant immunoglobulin libraries for specific- binding molecules (e.g. Orlandi R. et. al. (1989) PNAS 86:3833-3837, or Huse W.D. et. al. (1989) Science 256:1275-1281) or the in vitro stimulation of lymphocyte populations.
  • Current technology (Winter G. and Mistein C. (1991) Nature 349:293-299) provides a number of highly specific binding reagents based on the principles of antibody formation. These techniques may be adapted to produce molecules which specifically bind HEDGs
  • An additional embodiment of the subject invention is the use of HEDG specific antibodies, inhibitors, receptors or their analogs as bioactive agents to treat inflammation or disease including, but not limited to viral, bacterial or fungal infections; allergic responses; mechanical injury associated with trauma; hereditary diseases; lymphoma or carcinoma; or other conditions which activate the genes of lymphoid or neuronal tissues.
  • Bioactive compositions comprising agonists, antagonists, receptors or inhibitors of HEDG may be administered in a suitable therapeutic dose determined by any of several methodologies including clinical studies on mammalian species to determine maximal tolerable dose and on normal human subjects to determine safe dose. Additionally, the bioactive agent may be complexed with a variety of well established compounds or compositions which enhance stability or pharmacological properties such as half-life. It is contemplated that the therapeutic, bioactive composition may be delivered by intravenous infusion into the bloodstream or any other effective means which could be used for treating problems involving excessive lymphocyte and leukocyte trafficking.
  • HEDG Rheumatoid arthritis is currently evaluated on the basis of swelling, response to NSAIDs, x-rays, etc.
  • HEDG is most likely expressed on the surface of the fibroblasts, T and B lymphocytes, monocytelmacrophages, or mast cells which comprise the cells of the inflamed synovium.
  • an assay for the abnormal expression of HEDG is a viable diagnostic tool for assessing the extent that RA has progressed.
  • the expression of HEDG in a sustained inflammatory response makes it a valuable therapeutic target for screening drug libraries.
  • Inhibitors of HEDG are useful for controlling signal transduction and signaling cascades in cells of the rheumatoid synovium.
  • PCR reactions were conducted using EDG6-F1 or EDG6-R1 primers vs. primers derived from the pcDNA3 expression vector (Invitrogen catalog no. V790-20) in which this human kidney proximal tubule cDNA library was constructed (ATCC 87306).
  • EDG6-F1 5'-GGTGGTACTGCTCCTGGATGGTTTAG-3' (SEQ. ID NO:4)
  • EDG6-R1 5'-CGGAGGCACGCGCAGCAGAGAAGA-3' (SEQ. ID NO:5)
  • the one 3' PCR product, 3 '-2 contained the complete 3' portion of edg-6 coding sequence, including an extended 3 '-untranslated region and 34 bp poly(A) tail, characteristic of eukaryotic mRNAs.
  • a double digest was performed on 5 ⁇ l of the purified re-amplified PCR reaction as follows:
  • the restriction digest was incubated for 1 hour in a 37 ° C water block heater.
  • the reaction products were run on a gel, the ⁇ 2 kb DNA band was cut out and purified from the gel in 10 ⁇ l of l0 mM Tris (pH 8.5).
  • the double-digested, gel-purified PCR DNA was ligated into the prepared pcDNA3 plasmid vector using T4 DNA ligase kit (NEB, Cat #202CS) and transformed into Epicurean Coli XL-2 Blue MRF' Ultracompetent cells (Stratagene, Cat #200150). The transformation was plated onto 2xYT/ Ampicillin plates and single colonies were picked. DNA mini-preps were made using QIAGEN QIA-Prep 8 mini-prep kit (Cat #27144) and clones with appropriate inserts were identified by sequencing, carried out with the in-house ABI automated sequencing system. From this analysis, a full length clone designated pC3-hEdg6 was chosen for complete sequence determination of the cDNA insert.
  • An open reading frame of 1053 bp constitutes the coding region of human edg-6. This coding region starts with a methionine codon at nt 91 (figure 1, SEQ ID NO:l), preceded by an in-frame translation initiation codon (TGA) at nt 76-78. Translation is terminated by a stop codon at nt 1144.
  • GPCR G protein-coupled receptor
  • Intracellular loop- 1 (IL-1) :59-65
  • Extracellular loop- 1 (EL-1) : 95-108
  • a FASTA search against daily-updated in-house databases comprised of Genbank, EMBL, dbEST, the HTG, GSS and STS genomic sequencing databases revealed several sequence related to human edg-6.
  • Genbank W60555 the original EST (T02954) found by database mining, 1 additional EST (Genbank W60555) closely matched the edg-6 sequence (98.4% identity in a 386 nt overlap from nt 1078-1463).
  • the murine LPA receptor, edg-2 was the highest-scoring full-length cDNA sequence found from the combined Genbank/EMBL databases (Genbank MMU70622: 62.8% identity in 980 nt overlap from nt 106-1073). A lower degree of sequence identity was seen with the edg-1 orphan receptor (Genbank HUMEDG: 55.0% identity in 767 nt overlap from nt 277-1026).
  • Expression of hedg is accomplished by sub-cloning the cDNAs into appropriate expression vectors and transfecting the vectors into analogous expression hosts for example E.Coli.
  • the vector is engineered such that it contains a promoter for ⁇ -galactosidase, upstream of the cloning site, followed by sequence containing the amino-terminal Met and the subsequent 7 residues of ⁇ -galactosidase.
  • an engineered bacteriophage promoter useful for artificial priming and transcription and for providing a number of unique endonuclease restriction sites for cloning.
  • Induction of the isolated, transfected bacterial strain with IPTG using standard methods produces a fusion protein corresponding to the first seven residues of ⁇ - galactosidase, about 15 residues of "linker", and the peptide encoded within the cDNA.
  • cDNA clone inserts are generated by an essentially random process, there is one chance in three that the included cDNA will lie in the correct frame for proper translation. If the cDNA is not in the proper reading frame, it is obtained by deletion or insertion of the appropriate number of bases using well known methods including in vitro mutagenesis, digestion with exonuclease III or mung bean nuclease, or the inclusion of an oligonucleotide linker of appropriate length.
  • the hedg cDNA is shuttled into other vectors known to be useful for expression of protein in specific hosts.
  • Oligonucleotide primers containing cloning sites as well as a segment of DNA (about 25 bases) sufficient to hybridize to stretches at both ends of the target cDNA is synthesized chemically by standard methods. These primers are then used to amplify the desired gene segment by PCR. The resulting gene segment is digested with appropriate restriction enzymes under standard conditions and isolated by gel electrophoresis. Alternately, similar gene segments are produced by digestion of the cDNA with appropriate restriction enzymes. Using appropriate primers, segments of coding sequence from more than one gene are ligated together and cloned in appropriate vectors. It is possible to optimize expression by construction of such chimeric sequences.
  • Suitable expression hosts for such chimeric molecules include, but are not limited to, mammalian cells such as Chinese Hamster Ovary (CHO) and human 293 cells, insect cells such as Sf9 cells, yeast cells such as Saccharomyces cerevisiae, and bacteria such as E. coli.
  • a useful expression vector also includes an origin of replication to allow propagation in bacteria and a selectable marker such as the ⁇ -lactamase antibiotic resistance gene to allow plasmid selection in bacteria.
  • the vector may include a second selectable marker such as the neomycin phosphotransferase gene to allow selection in transfected eukaryotic host cells.
  • Vectors for use in eukaryotic expression hosts require RNA processing elements such as 3' polyadenylation sequences if such are not part of the cDNA of interest. Additionally, the vector contains promoters or enhancers which increase gene expression. Such promoters are host specific and include MMTV, SV40, and metallothionine promoters for CHO cells; trp, lac, tac and T7 promoters for bacterial hosts; and alpha factor, alcohol oxidase and PGH promoters for yeast. Transcription enhancers, such as the rous sarcoma virus enhancer, are used in mammalian host cells.
  • HEDG recombinantly produced HEDG are recovered from the conditioned medium and analyzed using chromatographic methods known in the art.
  • HEDG can be expressibly cloned into the expression vector pcDNA3, as exemplified herein.
  • This product can be used to transform, for example, HEK293 or COS by methodology standard in the art. Specifically, for example, using Lipofectamine (Gibco BRL catalog no. 18324-020) mediated gene transfer.
  • HEDG is expressed as a chimeric protein with one or more additional polypeptide domains added to facilitate protein purification.
  • purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle WA).
  • the inclusion of a cleaveable linker sequence such as Factor XA or enterokinase (Invitrogen) between the purification domain and the HEDG sequence is useful to facilitate expression of HEDG.
  • Functional chimeric T7Gs are constructed by combining the extracellular receptive sequences of a new isoform with the transmembrane and intracellular segments of a known isoform for test purposes. This concept was demonstrated by Kobilka et al (1988, Science 240:1310-1316) who created a series of chimeric ⁇ 2- ⁇ 2 adrenergic receptors (AR) by inserting progressively greater amounts of ⁇ 2-AR transmembrane sequence into ⁇ 2-AR. The binding activity of known agonists changed as the molecule shifted from having more ⁇ 2 than ⁇ 2 conformation, and intermediate constructs demonstrated mixed specificity.
  • T7G domain VII for ligand recognition was also found in chimeras utilizing two yeast ⁇ -factor receptors and is significant because the yeast receptors are classified as miscellaneous receptors. Thus, functional role of specific domains appears to be preserved throughout the T7G family regardless of category.
  • One heterologous system introduces genes for a mammalian T7G and a mammalian G-protein into yeast cells.
  • the T7G is shown to have appropriate ligand specificity and affinity and to trigger appropriate biological activation, growth arrest, and morphological changes of the yeast cells.
  • chimeric genes are created by combining sequences for extracellular receptive segments of any newly discovered T7G polypeptide with the nucleotides for the transmembrane and intracellular segments of the known P u molecule. Bathing the transfected K562 cells in microwells containing appropriate ligands triggers binding and fluorescent activity defining effectors of the T7G molecule. Once ligand and function are established, the P 2u system is useful for defining antagonists or inhibitors which block binding and prevent such fluorescent reactions.
  • denatured protein from reverse phase HPLC separation is obtained in quantities up to 75 mg. This denatured protein is used to immunize mice or rabbits using standard protocols; about 100 micrograms are adequate for immunization of a mouse, while up to 1 mg might be used to immunize a rabbit.
  • the denatured protein is radioiodinated and used to screen potential murine B-cell hybridomas for those which produce antibody. This procedure requires only small quantities of protein, such that 20 mg is sufficient for labeling and screening of several thousand clones.
  • the amino acid sequence of an appropriate HEDG domain is analyzed to determine regions of high antigenicity.
  • Oligopeptides comprising appropriate hydrophilic regions as illustrated in Figure 1, SEQ ID NO:2, are synthesized and used in suitable immunization protocols to raise antibodies. Analysis to select appropriate epitopes is described by Ausubel FM et al (supra).
  • the optimal amino acid sequences for immunization are usually at the C-terminus, the N-terminus and those intervening, hydrophilic regions of the polypeptide which are likely to be exposed to the external environment when the protein is in its natural conformation.
  • selected peptides about 15 residues in length, are synthesized using an
  • the resulting antisera are tested for antipeptide activity by binding the peptide to plastic, blocking with 1% bovine serum albumin, reacting with antisera, washing and reacting with labeled (radioactive or fluorescent), affinity purified, specific goat anti-rabbit IgG.
  • Hybridomas are prepared and screened using standard techniques. Hybridomas of interest are detected by screening with labeled HEDG to identify those fusions producing the monoclonal antibody with the desired specificity.
  • wells of plates FAST; Becton-Dickinson, Palo Alto CA
  • affinity purified, specific rabbit anti-mouse (or suitable antispecies Ig) antibodies at 10 mg/ml.
  • the coated wells are blocked with 1% BSA, washed and incubated with supematants from hybridomas. After washing the wells are incubated with labeled HEDG at 1 mg/ml.
  • Supematants with specific antibodies bind more labeled HEDG than is detectable in the background. Then clones producing specific antibodies are expanded and subjected to two cycles of cloning at limiting dilution. Cloned hybridomas are injected into pristane-treated mice to produce ascites, and monoclonal antibody is purified from mouse ascetic fluid by affinity chromatography on Protein A.
  • Monoclonal antibodies with affinities of at least 10 8 M- 1 , preferably 10 9 to 10 10 or stronger, are typically made by standard procedures as described in Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; and in Goding (1986) Monoclonal Antibodies: Principles and Practice, Academic Press, New York City, both incorporated herein by reference.
  • Particular HEDG antibodies are useful for investigating signal transduction and the diagnosis of infectious or hereditary conditions which are characterized by differences in the amount or distribution of HEDG or downstream products of an active signaling cascade.
  • Diagnostic tests for HEDG include methods utilizing antibody and a label to detect HEDG in human body fluids, membranes, cells, tissues or extracts of such.
  • the polypeptides and antibodies of the present invention are used with or without modification. Frequently, the polypeptides and antibodies are labeled by joining them, either covalently or non- covalently, with a substance which provides for a detectable signal.
  • labels and conjugation techniques are known and have been reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent agents, chemiluminescent agents, chromogenic agents, magnetic particles and the like. Patents teaching the use of such labels include US Patent No's.
  • HEDG soluble or membrane-bound HEDG
  • protocols for measuring soluble or membrane-bound HEDG are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescent activated cell sorting
  • a two-site monoclonal -based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on HEDG is preferred, but a competitive binding assay may be employed. These assays are described, among other places, in Maddox, DE et al (1983, J Exp. Med. 158:121 If).
  • Native or recombinant HEDG is purified by immunoaffinity chromatography using antibodies specific for HEDG.
  • an immunoaffinity column is constructed by covalently coupling the anti-TRH antibody to an activated chromatographic resin.
  • Polyclonal immunoglobulins are prepared from immune sera either by precipitation with ammonium sulfate or by purification on immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway NJ). Likewise, monoclonal antibodies are prepared from mouse ascites fluid by ammonium sulfate precipitation or chromatography on immobilized Protein A. Partially purified immunoglobulin is covalently attached to a chromatographic resin such as CnBr-activated Sepharose (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is blocked, and the derivative resin is washed according to the manufacturer's instructions.
  • a chromatographic resin such as CnBr-activated Sepharose
  • Such immunoaffinity columns are utilized in the purification of HEDG by preparing a fraction from cells containing HEDG in a soluble form. This preparation is derived by solubilization of whole cells or of a subcellular fraction obtained via differential centrifugation (with or without addition of detergent) or by other methods well known in the art. Alternatively, soluble HEDG containing a signal sequence is secreted in useful quantity into the medium in which the cells are grown.
  • a soluble HEDG-containing preparation is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of HEDG (e.g., high ionic strength buffers in the presence of detergent). Then, the column is eluted under conditions that disrupt antibody/protein binding (e.g., a buffer of pH 2-3 or a high concentration of a chaotrope such as urea or thiocyanate ion), and HEDG is collected.
  • a buffer of pH 2-3 or a high concentration of a chaotrope such as urea or thiocyanate ion
  • This invention is particularly useful for screening therapeutic compounds by using HEDG or binding fragments thereof in any of a variety of drug screening techniques.
  • HEDG is a G protein coupled receptor
  • any of the methods commonly used in the art may potentially used to identify HEDG ligands.
  • the polypeptide or fragment employed in such a test is either free in solution, affixed to a solid support, borne on a cell surface or located intracellularly.
  • One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or fragment. Drugs are screened against such transformed cells in competitive binding assays. Such cells, either in viable or fixed form, are used for standard binding assays.
  • One measures for example, the formation of complexes between HEDG and the agent being tested. Alternatively, one examines the diminution in complex formation between HEDG and a ligand caused by the agent being tested.
  • the present invention provides methods of screening for dmgs or any other agents which affect signal transduction. These methods, well known in the art, comprise contacting such an agent with HEDG polypeptide or a fragment thereof and assaying (i) for the presence of a complex between the agent and the HEDG polypeptide or fragment, or (ii) for the presence of a complex between the HEDG polypeptide or fragment and the cell. In such competitive binding assays, the HEDG polypeptide or fragment is typically labeled.
  • the invention is suitable for screening potential drugs by known methods of signal transduction assays for G protein coupled receptors.
  • the activity of a G protein coupled receptor such as HEDG can be measured using any of a variety of appropriate functional assays in which activation of the receptor results in an observable change in the level of some second messenger system, such as adenylate cyclase, guanylylcyclase, calcium mobilization, or inositol phospholipid hydrolysis.
  • One such method involves the following steps: a) co-transfection into a suitable cell of a plasmid including a reporter gene (for example, luciferase under the transcriptional control of SRE, serum response element) and an expression plasmid for HEDG.
  • SRE is known in the art as a common reporter for G protein coupled receptors which respond to mitogenic factors.
  • expression of HEDG c) pre-treatment of the transformed cell with serum starvation to reduce mitogenic signaling; d) application of LPA in serum free medium; and, e) after 5 hours, measuring luciferase activity.
  • This method relies on the activation of HEDG upon addition of LPA.
  • Expression of the reporter gene, coding for luciferase is induced by the mitogenic signal transduction resulting from the activation of HEDG by LPA.
  • other agonists of HEDG can be assayed. Therefore, with such a method, an increase in luciferase activity signals the presence of an agonist (i.e. LPA) for the subject receptor, while a decrease in luciferase activity signals the presence of an antagonist.
  • an luciferase is indicated as a preferred reporter in the above example, various other reporters (as discussed previously) can also be used.
  • the goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact, agonists, antagonists, or inhibitors. Any of these examples are used to fashion drugs which are more active or stable forms of the polypeptide or which enhance or interfere with the function of a polypeptide in vivo (e.g., Hodgson J. (1991) Bio/Technology 9:19-21, incorporated herein by reference).
  • the three-dimensional structure of a protein of interest, or of a protein-inhibitor complex is determined by x-ray crystallography, by computer modeling or, most typically, by a combination of the two approaches. Both the shape and charges of the polypeptide must be ascertained to elucidate the structure and to determine active site(s) of the molecule. Less often, useful information regarding the structure of a polypeptide is gained by modeling based on the structure of homologous proteins. In both cases, relevant structural information is used to design efficient inhibitors. Useful examples of rational dmg design includes molecules which have improved activity or stability as shown by Braxton S. and Wells J.A.
  • a target-specific antibody selected by functional assay, as described above, and then to solve its crystal structure.
  • This approach in principle, yields a pharmac ⁇ re upon which subsequent drug design is based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids is expected to be an analog of the original receptor. The anti-id is then used to identify and isolate peptides from banks of chemically or biologically produced peptides. The isolated peptides then act as the pharmacore.
  • anti-ids anti-idiotypic antibodies
  • the inventive purified HEDG is a research tool for identification, characterization and purification of interacting G or other signal transduction pathway proteins. Radioactive labels are inco ⁇ orated into a selected HEDG domain by various methods known in the art and used in vitro to capture interacting molecules. A preferred method involves labeling the primary amino groups in HEDG with 125 1 Bolton-Hunter reagent (Bolton, A.E. and Hunter, W.M. (1973) Biochem. J. 133: 529). This reagent has been used to label various molecules without concomitant loss of biological activity (Hebert CA. et. al. (1991) J. Biol. Chem. 266:18989: McColl S. et. al. (1993) J. Immunol. 150:4550-4555).
  • Labeled HEDG is useful as a reagent for the purification of molecules with which it interacts.
  • membrane-bound HEDG is covalently coupled to a chromatography column.
  • Cell-free extract derived from synovial cells or putative target cells is passed over the column, and molecules with appropriate affinity bind to HEDG.
  • HEDG-complex is recovered from the column, and the HEDG-binding ligand disassociated and subjected to N-terminal protein sequencing. This as sequence is then used to identify the captured molecule or to design degenerate oligonucleotide probes for cloning the relevant gene from an appropriate cDNA library.
  • antibodies are raised against HEDG, specifically monoclonal antibodies.
  • the monoclonal antibodies are screened to identify those which inhibit the binding of labeled HEDG. These monoclonal antibodies are then used therapeutically.
  • the subject primary sequence is isolated and subjected to any of the known methods for assaying protein interactions such as affinity columns, glass beads, or a two hybrid system (cDNA Library Protocols; Cowell, I.G. and Austin CA. (ed.), Methods in Molecular Biology, 69 (1996)).
  • LSTs are formulated in a nontoxic, inert, pharmaceutically acceptable aqueous carrier medium preferably at a pH of about 5 to 8, more preferably 6 to 8, although pH may vary according to the characteristics of the antibody, inhibitor, or antagonist being formulated and the condition to be treated. Characteristics of LSTs include solubility of the molecule, half-life and antigenicity/immunogenicity. These and other characteristics aid in defining an effective carrier. Native human proteins are preferred as LSTs, but organic or synthetic molecules resulting from drug screens are equally effective in particular situations.
  • LSTs are delivered by known routes of administration including but not limited to topical creams and gels; transmucosal spray and aerosol; transdermal patch and bandage; injectable, intravenous and lavage formulations; and orally administered liquids and pills particularly formulated to resist stomach acid and enzymes.
  • routes of administration including but not limited to topical creams and gels; transmucosal spray and aerosol; transdermal patch and bandage; injectable, intravenous and lavage formulations; and orally administered liquids and pills particularly formulated to resist stomach acid and enzymes.
  • the particular formulation, exact dosage, and route of administration is determined by the attending physician and varies according to each specific situation. Such determinations are made by considering multiple variables such as the condition to be treated, the LST to be administered, and the pharmacokinetic profile of a particular LST.
  • LST formulations might be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular LST. Normal dosage amounts vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature; see US Patent Nos. 4,657,760; 5,206,344; or 5,225,212. Those skilled in the art employ different formulations for different LSTs. Administration to cells such as nerve cells necessitates delivery in a manner different from that to other cells such as vascular endothelial cells.
  • abnormal signal transduction, trauma, or diseases which trigger HEDG activity are treatable with LSTs. These conditions or diseases are specifically diagnosed by the tests discussed above, and such testing should be performed in suspected cases of viral, bacterial or fungal infections: allergic responses; mechanical injury associated with trauma; hereditary diseases; lymphoma or carcinoma; or other conditions which activate the genes of lymphoid or neuronal tissues.
  • Autoimmune disorders whereby antibodies are produced against HEDG can be expected to be associated with disease states.
  • diseases can be expected to be associated with decreased muscle activity that presents symptoms much like myasthenia gravis, or to be associated with decreased pain perception.
  • a disease caused by autoantibodies to a molecule involved in neurotransmission glutamic acid decarboxylase
  • Nathan et al. J. Neurosci. Res. 40: 134-137, 1995.
  • the presence of these antibodies can be measured by established immunological methods using protein sequences obtained from the nucleic acids described herein or the related glycine transporters reported elsewhere. See, for example, Kim et al., Mol. Pharmacol., 45: 608-617, 1994 and Liu et al., J. Bio. Chem. 268: 22802-22808, 1992. Such immunological methods are described, for example, in Ausubel et al., Short Protocols in Molecular Biology, John Wiley & Sons, New York, 1992. EXAMPLE 15
  • Animal model systems which elucidate the physiological and behavioral roles of the HEDG receptor are produced by creating transgenic animals in which the activity of the HEDG receptor is either increased or decreased, or the amino acid sequence of the expressed HEDG receptor is altered, by a variety of techniques.
  • these techniques include, but are not limited to: 1) Insertion of normal or mutant versions of DNA encoding a HEDG receptor, by microinjection, electroporation, retroviral transfection or other means well known to those skilled in the art, into appropriate fertilized embryos in order to produce a transgenic animal or 2) Homologous recombination of mutant or normal, human or animal versions of these genes with the native gene locus in transgenic animals to alter the regulation of expression or the structure of these HEDG receptor sequences.
  • the technique of homologous recombination is well known in the art. It replaces the native gene with the inserted gene and so is useful for producing an animal that cannot express native HEDG receptors but does express, for example, an inserted mutant HEDG receptor, which has replaced the native HEDG receptor in the animal's genome by recombination, resulting in underexpression of the transporter. Microinjection adds genes to the genome, but does not remove them, and so is useful for producing an animal which expresses its own and added HEDG receptors, resulting in overexpression of the HEDG receptors.
  • One means available for producing a transgenic animal is as follows: Female mice are mated, and the resulting fertilized eggs are dissected out of their receptor is cesium chloride oviducts. The eggs are stored in an appropriate medium such as M2 medium. DNA or cDNA encoding a HEDG purified from a vector by methods well known in the art. Inducible promoters may be fused with the coding region of the DNA to provide an experimental means to regulate expression of the transgene. Alternatively or in addition, tissue specific regulatory elements may be fused with the coding region to permit tissue-specific expression of the trans-gene.
  • microinjection needle which may be made from capillary tubing using a piper puller
  • the egg to be injected is put in a depression slide.
  • the needle is inserted into the pronucleus of the egg, and the DNA solution is injected.
  • the injected egg is then transferred into the oviduct of a pseudopregnant mouse (a mouse stimulated by the appropriate hormones to maintain pregnancy but which is not actually pregnant), where it proceeds to the uterus, implants, and develops to term.
  • pseudopregnant mouse a mouse stimulated by the appropriate hormones to maintain pregnancy but which is not actually pregnant
  • (l) APPLICANT MUNROE, Donald G. VYAS, Tejal B.
  • CACACTGGTC CCCGCACAGC CCGACTTTCA CTTGAGGGCT GGTTCCTGCG GCAGGGCTTG 420
  • GGAGGTGCCA GCACTCGCAT CATGCTTCCC GAGAACGGCC ACCCACTGAT GGACTCCACC 1140
  • GAAGCAGTCC ACGGGGAGGG GATGATACAA GGAGTAAACC TTTCTTTACA CTCTGAGGTC 1740
  • CTGTCCACTA TACATCCTCT GCCCAGGGAG GTGCCAGCAC TCGCATCATG CTTCCCGAGA 1200
  • MOLECULE TYPE DNA
  • SEQUENCE DESCRIPTION SEQ ID NO: 6: TAGAGAACCC ACTGCTTAC 19

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Abstract

L'invention concerne une séquence d'acide nucléique isolée, qui code une séquence d'acides aminés destinée à un nouvel homologue du récepteur EDG-6 humain. Elle concerne également des polypeptides du récepteur EDG-6 humain purifiés, dérivés dudit acide nucléique, ainsi que des méthodes et des animaux transgéniques pour lesdits dérivés.
PCT/CA1998/000487 1997-05-22 1998-05-22 Homologue du recepteur edg-6 humain WO1998053062A1 (fr)

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EP0855443A2 (fr) * 1997-01-28 1998-07-29 Smithkline Beecham Corporation Clone de cADN HE8CH90 qui code pour un récepteur 7-transmembranaire
WO1999035259A1 (fr) * 1997-12-30 1999-07-15 Nps Allelix Corp. Identification des recepteurs des lysolipides impliques dans la reponse inflammatoire
EP1173880A1 (fr) * 1999-03-23 2002-01-23 The Regents Of The University Of California Recepteurs de polypeptides humains pour lysophospholipides et sphingolipides et acides nucleiques les codant
US6733990B1 (en) 1999-08-03 2004-05-11 Millennium Pharmaceuticals, Inc. Nucleic acid encoding 15571, a GPCR-like molecule of the secretin-like family

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EP1597581A1 (fr) * 2002-11-13 2005-11-23 Bayer HealthCare AG Outils de diagnostics et therapeutique pour maladies associees a la differenciation endotheliale, recepteur 4 couple aux proteines g (edg4) sphingolipidique
CA2506243A1 (fr) * 2002-11-19 2004-06-03 Amgen Inc. Genes amplifies impliques dans un cancer

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WO1997000952A2 (fr) * 1995-06-20 1997-01-09 Incyte Pharmaceuticals, Inc. Homologue du recepteur de edg-2 de l'homme
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HECHT J H ET AL: "VENTRICULAR ZONE GENE-1 (VZG-1) ENCODES A LYSOPHOSPHATIDIC ACID RECEPTOR EXPRESSED IN NEUROGENIC REGIONS OF THE DEVELOPING CEREBRALCORTEX", THE JOURNAL OF CELL BIOLOGY, vol. 135, no. 4, November 1996 (1996-11-01), pages 1071 - 1083, XP002046888 *
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Cited By (9)

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Publication number Priority date Publication date Assignee Title
EP0855443A2 (fr) * 1997-01-28 1998-07-29 Smithkline Beecham Corporation Clone de cADN HE8CH90 qui code pour un récepteur 7-transmembranaire
EP0855443A3 (fr) * 1997-01-28 2000-03-01 Smithkline Beecham Corporation Clone de cADN HE8CH90 qui code pour un récepteur 7-transmembranaire
WO1999035259A1 (fr) * 1997-12-30 1999-07-15 Nps Allelix Corp. Identification des recepteurs des lysolipides impliques dans la reponse inflammatoire
US6482609B1 (en) 1997-12-30 2002-11-19 Nps Allelix Corporation Isolated human EDG-4 receptor and polynucletide encoding said receptor
US7052850B2 (en) 1997-12-30 2006-05-30 Nps Allelix Corp. Isolated human EDG-4 receptor
EP1173880A1 (fr) * 1999-03-23 2002-01-23 The Regents Of The University Of California Recepteurs de polypeptides humains pour lysophospholipides et sphingolipides et acides nucleiques les codant
US6812335B1 (en) 1999-03-23 2004-11-02 The Regents Of The University Of California Human polypeptide receptors for lysophospholipids and sphingolipids and nucleic acids encoding the same
EP1173880A4 (fr) * 1999-03-23 2005-02-16 Univ California Recepteurs de polypeptides humains pour lysophospholipides et sphingolipides et acides nucleiques les codant
US6733990B1 (en) 1999-08-03 2004-05-11 Millennium Pharmaceuticals, Inc. Nucleic acid encoding 15571, a GPCR-like molecule of the secretin-like family

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